DE3877197T2 - METHOD FOR PRODUCING HIRUDINE. - Google Patents

Description

Summary of the invention

The present invention relates to a new process for producing hirudin. Hirudin can be defined as a protease inhibitor with antithrombin activity, which is present in the salivary organs (sialades) of Hirudo medicinalis. Unlike ordinary antithrombin drugs, the hirudin of the present invention has a direct inhibitory effect on thrombin.

For this reason, hirudin is expected to act as a preventive or antidote for thrombosis.

In conventional techniques, the small amount of hirudin is only extracted directly from Hirudo medicinalis. A technique is already known in which hirudin is produced in E. coli, but the amount of hirudin accumulated in it is so small that the technique is considered not useful in the medical field.

In the present invention, the use of genetic engineering allows hirudin to be produced in dramatically large quantities.

Short description of the drawings

Fig. 1 is a view showing an assembly process of a vector pNPA225 for secretion and expression of a heterologous protein used in Examples.

Fig.2 is a view showing an assembly process of a hirudin secretion vector pNPH208 used in the Examples.

Fig.3 is a view showing a synthesized oligonucleotide for encoding amino acids from the N-terminus to the tenth amino acid of hirudin.

Fig. 4 is a view showing an assembly process of a hybrid plasmid p3009 containing the base sequence for encoding hirudin.

Here, symbols are explained in connection with the drawings: "promoter" denotes the promoter region of a neutral protease gene; "SD" a ribosome binding region within a neutral protease gene; "pre" a region encoding a secretion signal of a neutral protease gene; "Δpro" an upstream region of a propeptide of the neutral protease gene; "α-amylase" a DNA sequence encoding α-amylase; "H" a DNA sequence encoding hirudin; "ΔH" an upstream region of a DNA sequence encoding hirudin; and "Δ protease" a back portion of a neutral protease gene.

Detailed description of the invention

A hirudin produced in the present invention acts directly on thrombin in blood and can therefore be used for the medical treatment of thrombosis.

Hirudin is a protease inhibitor with antithrombin activity, which is present in salivary organs of Hirudo medincinalis, which is a kind of eukaryote. Furthermore, hirudin does not comprise a single protein, but a group of different types of molecules. For example, HVI (Reference 1) and HV2 (Reference 2) are known to be part of a molecule that forms hirudin, and primary structures of them have also been reported (References 1 and 2).

These primary structures have high homology and common structural features of these hirudin molecules are that a tyrosine residue in the form of a sulfate monoester is present in them and that three intramolecular S-S bonds are present in them.

Generally, thrombin is present in blood as inactive prothrombin and is activated according to the request of the organism to convert fibrinogen into fibrin. In short, thrombin takes charge of an important biological mechanism in blood coagulation system. If thrombin is pathologically activated in the whole blood of an organism, fibrin is deposited in a microvascular system so that thrombin or thrombi are formed therein. The thrombus can be defined as a mass of coagulated blood in the blood vessel and the pathological phenomenon of formation of such thrombin or thrombi is called thrombosis. The thrombosis leads to narrowing and obstruction of blood vessels due to the thrombin or thrombi so that ischemic pathology or infarction takes place in major organs such as heart, cerebrum and lungs to cause their functional disorders. In previous years, great attention has been paid to thrombosis because the latter deals with the occurrence of organ pathology attributable to immunological disorder such as nephritis or pneumonia, and it also deals with concomitant pathology at the time of organ transplantation or exchanged blood vessels. In addition, attention is also paid to diffuse intravascular coagulation syndrome (DIC), which is known as a specific pathological symptom in which thrombi frequently occur in a microvascular system. The report concerning DIC was made in 1960, and DIC was first considered as a rare syndrome. However, later it became apparent that DIC is not rare, and with reference to various clinical symptoms which were considered as bleeding at the end of various diseases or as organ pathologies without sufficient investigation, it became clear that these clinical symptoms result from DIC.

Nowadays, thrombosis has an increasing tendency. It is known that the thrombus is often formed at a position on the inner wall of a blood vessel where an abnormal change, especially sclerosis or inflammation, is present, and such pathology increases with age. Furthermore, the worldwide prolongation of life is a cause for the increase in thrombosis.

Clinical pathological examples of thrombosis include: (stroke) attack, myocardial infarction, deep vein thrombosis, obstruction of arteries of the extremities, pulmonary thrombosis and retinal artery thrombosis. The total sum of thrombosis in various organs ranks at the top of all diseases in terms of disease incidence rate and cause of death. In the future, therefore, the clinical and pathological significance of thrombosis will become increasingly important.

As remedies for the thrombosis just described, heparin, which acts through antithrombin III, and antivitamin K, which inhibits the biosynthesis of a vitamin K-dependent blood coagulation factor, are known. In addition, another thrombin inhibitor is gabexate mesylate, which is a proteolytic enzyme inhibitor.

It is previously well known that heparin is an antithrombin agent that is widely used for thrombosis including DIC. However, the function of heparin is to accelerate the inhibitory effect of antithrombin III on blood coagulation and therefore heparin is not considered effective for thrombosis associated with DIC or nephrosis where the content of antithrombin III is low (Reference 3). In addition, the gabexate mesylate agent has an inhibitory effect on physiologically important enzymes such as plasmin, kallikrein and trypsin. Accordingly, great caution is required when using the gabexate mesylate agent.

Under such circumstances, the development of a new preventive or antidote for thrombosis including DIC is important for therapeutic and preventive medicine.

On the other hand, hirudin has a direct inhibitory activity on thrombin, unlike common agents for thrombosis. In particular, hirudin has a high functional specificity for thrombin and prethrombin 2 (dissociation constant = 0.8 x 10⁻¹⁰) (Reference 4), and it inhibits only activator No. IV alone in addition to thrombin. That is, hirudin never inhibits enzymes other than those affecting blood coagulation. Moreover, the toxicity of hirudin is extremely low and it is called non-antigenic, and hirudin, which has biological activity, is rapidly excreted by the kidneys into the urine (Reference 5). Thus, hirudin can can be used instead of conventional antithrombic agents as a useful preventive and antidote for thrombosis including DIC.

Before recombinant DNA technology was developed, hirudin was directly extracted from Hirudo medicinalis. However, even to obtain the small amount of hirudin in this way, a large amount of fasted Hirudo medicinalis was necessary, and even when crude hirudin was prepared, a complicated purification step and much time were required. For example, even in the case that crude hirudin with a purity of about 10% was prepared, in which impure proteins derived from Hirudo medicinalis are present, a homogenate of Hirudo medicinalis fasted for 2 to 3 weeks is first subjected to hot water extraction, and then to ethanol precipitation, fractional precipitation with acetone, adsorption and desorption using bentonite, and finally precipitation at the isoelectric point must be carried out successively. Furthermore, the precipitation of pure hirudin from the crude product requires ECTEOLA cellulose column chromatography, Sephadex CM C-25 column chromatography and gel filtration using Sephadex G-25, and according to a report, in such a case the yield is less than 0.001% (Reference Literature 6). Since it is impossible to obtain hirudin in the amounts described above, this ingredient cannot now be used in practice as a medicine and the therapeutic use of hirudin, which can be expected from its superior characteristics, has not been achieved so far. For this reason, it is strongly demanded to establish an efficient manufacturing process for hirudin.

In the near future, mass production of a heterologous gene product will be possible using recombinant DNA technology, in which a heterologous gene is expressed in a microorganism host. The heterologous gene product from the microorganism can be defined as a protein that results from the expression of a gene that does not originate from this microorganism.

The production of a desired substance by using recombinant DNA technology, in which a microorganism is used as a host, can be roughly divided into intracellular production and extrabacterial secretory production.

In the earlier preparation, the heterologous gene product can be effectively maintained in bacteria, but the following problems arise:

In the first place, the heterologous gene product obtained in bacteria is usually an insoluble mass called inclusion body without biological activity. In general, it is not difficult to recover such an inclusion body from the bacteria. However, in order to obtain the heterologous gene product with a natural higher order structure and activity, the inclusion body must be made soluble by using a solubilizing agent such as urea and after that it is necessary for the inclusion body to return to the heterologous gene product with the natural higher order structure and activity. This method is considered impractical because it is very complicated and its efficiency is low. For example, according to Schoner et al. (Reference 7), the inclusion body of bovine growth hormone formed in E. coli is partially solubilized in the presence of 5 moles of urea, but the amount of solubilized inclusion body is only 10% of the total amount and the rest of it remains in the state of pellets.

Second, it is difficult to obtain a natural heterologous gene product.

Usually, in the case where the heterologous gene is expressed by using a microorganism, it is necessary that an initiation codon (ATG) exists at the 5'-terminus of the gene encoding a protein. Consequently, a fused protein with Met attached to the N-terminus is produced. If such a fused protein with Met attached is administered to a human as a medicine, the occurrence of an antibody based on the fused protein is increased, so that side effects such as deterioration of medicinal efficacy and anaphylaxis take place inconveniently. In fact, a human growth hormone (hGH) produced in E. coli was a Met-hGH with Met attached to the N-terminus and had approximately the same effects as natural hGH. However, it was observed that the incidence of antibody was high when Met-hGH was administered to a human (Reference 8).

Third, it was also known that when the heterologous gene product with S-S bonds was produced in bacteria, the S-S bonds were not formed exactly. For example, it was reported by J.M. Schoemarker (Reference Example 9) that when a bovine chymosin (rennet) with three S-S bonds in one molecule was expressed by E. coli, the S-S bond was formed between the molecules.

Also with regard to hirudin, there is a known state of the art (Reference 10) for its production in E. coli, but in this case, it has been reported in the literature that the hirudin accumulated in the bacteria has only an antithrombin activity corresponding to 0.2 mg/L x A600. The reason why the amount of accumulated hirudin is so low could be that in E. coli the inactive hirudin is accumulated, in which the S-S bond, which is essential for the expression of the antithrombin activity, is not precisely formed.

Although a method for the formation of hirudin with high antithrombin activity is now required, the intrabacterial production method is not desirable for the reasons mentioned above.

On the other hand, the extrabacterial secretory production process for the useful heterologous gene product entails various problems to be solved, but nevertheless this process is considered more suitable for the purpose of the present invention.

Generally, a secretory protein in bacteria is synthesized as a precursor protein in which a secretion signal is attached to the N-terminus of the mature protein, and the secretion signal of the precursor protein is removed therefrom in the course of secretion, so that the mature protein is released from the bacteria without a secretion signal (Reference 11). Here, the mature protein mentioned above can be defined as a secretion signal-free secretory protein.

If hirudin is expressed in bacteria as a precursor protein in which the secretion signal is present at the N-terminus of hirudin and then secreted by the bacteria, the hirudin thus obtained can have antithrombin activity, thereby solving the above-mentioned problem of the extracellular secretory production process. In addition, if the ligation manner of the secretion signal to hirudin is successfully accomplished, the secretion signal at a desired site in hirudin can be cleaved at the time of secretion, and a hirudin having the same N-terminus as in hirudo medicinalis-derived hirudin can be expected to be produced.

Now, the present inventors have investigated methods of using bacteria of the Bacillus genus as a host, which have the feature of secreting a large amount of secretory protein and are non-pathogenic and are widely used as industrial microorganisms for enzymes, amino acids, nucleic acids and the like. Particularly with respect to Bacillus subtilis of the Bacillus genus, there are abundant data from the viewpoint of genetics, biochemistry, molecular biology and applied microbiology. Thus, it is important to introduce a host-vector system using the Bacillus subtilis host in which the heterologous gene product is formed in Bacillus subtilis and then released from the bacteria.

Previously, reports were made on the secretion of β-lactamase from E. coli using the α-amylase gene of Bacillus amyloliquefaciens (Reference 12), the secretion of nuclease from Staphylococcus aureus (Reference 13), the secretion of protein A from Staphylococcus aureus (Reference 14), the secretion of human α-interferon (Reference 15), the secretion of mouse β-interferon by using an α-amylase gene from Bacillus subtilis (Reference 16), the secretion of protein A from Staphylococcus by using a neutral protease gene or an alkaline protease gene from Bacillus amyloliquefaciens (Reference 17), and the secretion of human serum albumin (Reference 18). According to these reports, when Bacillus subtilis is used as a host, a protein derived from a certain type of prokaryote can be secreted and efficiently accumulated.

For example, the amounts of secreted and accumulated β-lactamase (Reference 12), nuclease (Reference 13), protein A (Reference 17) are approximately 20 mg, approximately 50 mg or approximately 1000 mg per liter of culture medium.

However, the above-mentioned reports indicate that the secreted amounts of human α-interferon (Reference 15), mouse β-interferon (Reference 16), human serum albumin (Reference 18) derived from eukaryotes are very small. For example, there is a report that the amount of human α-interferon is 0.5 mg per liter of culture medium. As shown in these reports, it is not always easy to obtain a large amount of a protein derived from a eukaryote by using Bacillus subtilis as a host.

Generally, the gene of the secreted protein is transcribed into mRNA by RNA polymerase, and this mRNA is used as a template to synthesize a precursor protein in which a secretion signal is added to the N-terminus of the mature protein. It is known that the secretion signal is removed from the precursor protein during the course of secretion.

For the purpose of finding out a reason why the amount of secreted and accumulated protein depends on the type of protein itself, Ulmanen et al. (Reference 19) studied the transcription and translation of a fused gene in which a DNA sequence encoding a heteroprotein was ligated to a site immediately preceding the region encoding the promoter, ribosome binding region and secretion signal of a secretion protein gene, and further studied a secretion efficiency of the resulting heterologous gene product to determine which of the above-mentioned factors influences the amount of secreted and accumulated heterologous gene product.

That is, they tested the secretion of EI protein, which is the glycoprotein of Semiliki Forest Virus, β-lactamase of E. coli and α-amylase gene of Bacillus amyloliquefaciens by using the region coding for a promoter, a ribosome binding region and the secretion signal of the amylase gene of Bacillus amyloliquefaciens. As a result, it was seen that the amount of mRNA coding for the precursor protein in which the secretion signal was added to the site above the N-terminus of a mature protein produced in bacteria changes terribly.

On the other hand, it has been reported that the amounts of secreted and accumulated E1 protein and β-lactamase are 0.01% and 10%, respectively, of the amount of secreted and accumulated α-amylase. According to this report, the reason for the above-mentioned results is that Bacillus subtilis cannot efficiently secrete certain kinds of fused protein in which a DNA sequence for a heterologous protein is fused to the 3'-terminus of the secretion signal coding region of the α-amylase gene, or the reason is the decomposition of the heterologous protein by the proteolytic activity of the host after or during secretion.

As can be understood from the foregoing, it has been reported that even when the same secretion protein gene, i.e., the same region encoding the promoter, ribosome binding region and secretion signal of the α-amylase gene, has been used, the amount of protein secreted and accumulated depends on the type of protein itself.

According to the report by Palva I (Reference 15) and Schein CH (Reference 20), even if the same region coding for the secretion signal of a secretory protein gene and the same DNA fragment coding for a protein derived from a eukaryote have been used, a difference in ligation structure between the two, so that in one case, the protein derived from the eukaryote is secreted by Bacillus subtilis or in another case it is not secreted by it.

Palva et al. reported the following experiment: A DNA fragment encoding a mature interferon (IFN) is ligated, either directly or through a 5-amino acid residue junction region (Asn-Gly-Thr-Glu-Ala), to a site downstream of the region (Ala-Val) containing a cleavage site of the secretion signal in the α-amylase gene of Bacillus amyloliquefaciens, which is one of the secretion proteins, i.e., a DNA fragment containing the region (Val) encoding an N-terminal amino acid of a mature protein at the downstream side of the 3'-terminus encoding the secretion signal. In this case, the secretion signal is removed, but the secreted and accumulated interferon is a fused protein with an amino acid (Val) added to the N-terminus of the mature interferon or it is another fused protein with 6 amino acids (Val-Asn-Gly-Thr-Gln-Ala) added, and the total amount of these interferons is 0.5 mg to 1 mg per liter of culture medium. On the other hand, Schein et al. have attempted the secretion of a human IFN by ligating the DNA fragment coding for the mature interferon to a site immediately after the region coding for the amino acid (Ala) of the C-terminus of the secretion signal of the same α-amylase gene as in Palva et al. However, in this case, it was reported that a large amount of precursor IFN or mature IFN remained in the bacterial cells and the efficiency of secretion into the culture medium was very low.

As discussed above, it is unpredictable whether or not the precursor in which the secretion signal is located at a site upstream of the N-terminus of the eukaryotic-derived mature protein is secreted into a system using Bacillus subtilis as a host and whether or not the secretion signal is processed during secretion. It is widely accepted that these results depend on the combination of the mature protein and secretion signal selected or on the manner in which the two are ligated.

Under these circumstances, the present invention has been completed, and an object of the present invention is to provide hirudin which is a useful antithrombic agent as a preventive and antidote for thrombosis, and other objects in the present case are to provide a hirudin secretion plasmid, to provide a transformed strain, and to provide a production process for hirudin using the transformed strain.

Taking the above-described situations into consideration, the present inventors have conducted research to search for a gene having a promoter, a ribosome binding region and a secretion signal which allows the secretion of hirudin and as a result, the present invention was finally completed.

That is, the present invention is directed to a hirudin secretion plasmid in which a DNA sequence is ligated into a vector DNA, the aforementioned DNA sequence having been prepared by ligating a DNA fragment coding for hirudin to a site immediately after the region coding for the promoter, ribosome binding region and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens; to a transformed strain obtained by transformation with the above-mentioned hirudin secretion plasmid, and to a method for producing hirudin comprising the steps of culturing the above-mentioned transformed strain and recovery of hirudin from the culture medium supernatant.

Now, the present invention will be described in detail.

The promoter referred to in the present invention can be defined as a DNA sequence which is recognized by an RNA polymerase and to which the latter binds.

It is known that when the initiation point of RNA synthesis is considered as "+1", usually two consensus DNA sequences are present in the -35 region (5'-TTGACA-3') and -10 region (5'-TATAAT-3'). In general, the "-35 region" is considered necessary for the recognition of RNA polymerase and the "-10 region" is considered necessary for the ligation of RNA polymerase (Reference 21).

As is already known, Bacillus subtilis possesses various types of RNA polymerases. This versatility plays an important role in the process of sporulation, which includes the difficult expression control of Bacillus subtilis. In particular, most polymerases in a vegetative propagation phase include a 55-type RNA polymerase and therefore it is known that the transcription of most genes is carried out by this type of polymerase (Reference 22).

The DNA sequence of the present invention in which the DNA fragment encoding hirudin has been ligated to a site immediately after the region encoding the promoter, ribosome binding site and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens is as follows:

In this DNA sequence, it is assumed that the "-10 region" of the promoter is 5'TATTAT3' (b-part of the above sequence) and the "-35 region" is 5'TTGCAG3' (a-part of the above sequence).

This DNA sequence has a high homology to the consensus sequence recognized by RNA polymerase type 55, which is the predominant RNA polymerase in the vegetative propagation phase of Bacillus subtilis (Reference 22).

Furthermore, the ribosome binding region can be defined as the DNA sequence encoding a region of mRNA to which ribosome mRNA can be bound that has been synthesized by the RNA polymerase, whereby the mRNA combines with a ribosome.

In general, the ribosome binding region is a DNA sequence present 5 to 9 bases upstream of the initiation codon, which is complementary to the 3'-terminal sequence of the 16S RNA initiation codon and which is complementary to that of a 16S rRNA 3'-terminus. The DNA sequence of the 16S rRNA depends on the type of microorganism, but it is known that the DNA sequence of the 16S rRNA of Bacillus subtilis is 3'UCUUUCCUCC5' (Reference 22).

As the DNA sequence considered as the ribosome binding region in the above-mentioned DNA sequence, there is a sequence 5'AAAGGGG3'(c-part in the sequence shown above), and this DNA sequence has extremely high complementarity to the 16S rRNA of Bacillus subtilis.

The DNA sequences of the promoter and the ribosome binding region just described play an important role in the expression of the gene. Today it is widely known that these DNA sequences are involved in the expression efficiency of a gene (Reference 21).

In the case where the gene of a desired protein is expressed using a Bacillus bacterium as a host, the RNA polymerase and ribosome of a Bacillus bacterium have strict specificity for the promoter and ribosome binding region (Reference 22), and therefore the regions therefor are desirably derived from the Bacillus bacterium (Reference 23).

The secretion signal can be defined as a polypeptide with 20 to 30 amino acid residues present preceding the N-terminus of a mature protein. Characteristics of the secretion signal are as follows: it is known that a basic amino acid is present near the N-terminus, that the cluster of a hydrophobic amino acid exists in the middle of it, and that an amino acid with a small side chain is present at the cleavage site of the secretion signal. This polypeptide is removed in the course of secretion and is considered to play an important role in the passage of a precursor protein through a cytoplasmic membrane (Reference 11).

The amino acid sequence which is considered as the secretion signal of the neutral protease gene of Bacillus amyloliquefaciens and used for the assembly of the hirudin secretion plasmid according to the present invention is Met-Gly-Leu-Gly-Lys-Lys-Leu-Ser-Ser-Ala-Val-Ala-Ala-Ser-Phe-Met-Ser-Leu-Thr-Ile-Ser-Leu-Pro-Gly-Val-Gln-Ala-, and this sequence has a typical primary structure of the secretion signal. That is, in this amino acid sequence, it can be understood that Lys, which is a basic amino acid, is present in the vicinity of the N-terminus, that the cluster (Leu-Ser-Ser-Ala-Val-Ala-Ala-Ser-Phe-Met-Ser-Leu-Thr-Ile-Ser-Leu) of a hydrophobic amino acid exists in the middle of it, and that an amino acid (Ala) with a small side chain is present at the cleavage site of the secretion signal.

Thus, the DNA sequence encoding the secretion signal in the above-mentioned DNA sequence is:

(d-part in the DNA sequence mentioned above).

The amino acid sequence of the hirudin protein used for the assembly of the hirudin secretion plasmid of the present invention is: and the DNA sequence for hirudin is:

(e-part of the DNA sequence mentioned above).

In recent years, the DNA sequences of many genes have been clarified and it is possible to investigate the frequency of codons used in the genes. As a result, it has been seen that the frequency of codons used depends on the kind of creature. Thus, in the case that the DNA sequence is chemically synthesized in order to obtain highly efficient expression, the usual way is to design the DNA sequence so that suitable codons can be sufficiently contained therein. In assembling the hirudin secretion vector of the present invention, the DNA sequence coding for chemically synthesized hirudin is used. Usually, a DNA sequence coding for a polypeptide is not limited to one kind of codon and therefore various DNA sequences coding for hirudin can be devised. However, in the present invention, the DNA sequence designed to contain many desirable codons for Bacillus subtilis is used, considering that the expression is carried out using Bacillus subtilis as a host.

For the secretion of a heterologous protein by Bacillus subtilis, it is essential that the heterologous gene encoding the desired protein has been ligated to the region encoding the promoter, the ribosome binding region and the secretion signal, which is derived from a gene for an extracellular protein. It is important to use the gene for the extracellular protein which can be produced extracellularly in large quantities and in a short time.

As such secretion proteins, alkaline phosphatase, α-amylase, levansucrase or levaninvertase and the like are known. However, it should be noted that the heterologous gene product cannot be efficiently secreted in time only by assembling the secretion plasmid for a heterologous protein in which the DNA fragment coding for the heterologous protein has been ligated to a site following the region coding for the promoter, ribosome binding region and secretion signal of such a secretion protein and can be replicated with Bacillus subtilis. This fact has been reported by Ullmanen et al. (Reference 19) and Schein (Reference 20).

The inventors of the present application have intensively researched to obtain the gene coding for the secretion protein having the ability by which hirudin can be secreted in large quantities from the bacterium, and have also researched a ligation manner between the DNA fragment coding for the secretion signal and the DNA fragment coding for hirudin. As a result, it has been found that it is possible to assemble the secretion plasmid of the present invention which provides such extremely good results as shown in the examples of the present case when the neutral protease gene of a Bacillus bacterium, particularly preferably Bacillus amyloliquefaciens, is selected and when the DNA fragment encoding hirudin has been ligated to a site immediately after the region encoding the promoter, the ribosome binding region and the secretion signal.

As the vector DNA for forming the hirudin secretion plasmid of the present invention, any can be used as long as it can be replicated in Bacillus subtilis. Examples of the vector that can be commonly used include pUB110, pTP5, pC194, pDB9, pBD64, pBC16 and pE194, which are plasmids derived from Staphylococcus. Bacillus subtilis having the above-mentioned plasmids can be available to anyone at the Bacillus Stock Center in Ohio University (address: 484 West 12th Avenue, Columbus, Ohio 43210 USA).

In particular, as the vector DNA used in the present invention, any can be used as long as it is a plasmid that can be replicated in Bacillus bacteria. However, pUB110 is the best because molecular biological data on it are abundant and it can be stably maintained in Bacillus bacteria.

As described in the examples given below, the hirudin secretion plasmid of the present invention can be prepared by ligating the DNA fragment encoding hirudin to a site immediately after the region encoding the promoter, ribosome binding region and secretion signal of a neutral protease gene by using a heterologous protein expression vector capable of encoding a heterologous protein immediately after the region encoding the above promoter, ribosome binding region and secretion signal. The hirudin secretion plasmid of the present invention can be assembled by combining a chemically synthesized DNA fragment containing the above-mentioned DNA sequence with a suitable restriction enzyme cleaved vector DNA fragment which can be replicated in Bacillus subtilis according to a conventional ligation technique. In these cases, two DNA fragments can be ligated together, for example via a common restriction enzyme site and/or by using a synthesized DNA linker and/or by means of a blunt-end ligation.

Bacillus subtilis can be transformed to obtain a transformed strain by using the hirudin secretion plasmid assembled by ligating the DNA fragment into the above-mentioned vector DNA, the aforementioned DNA fragment being prepared by ligating the DNA fragment encoding the hirudin protein to a site after the region encoding the promoter, ribosome binding region and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens.

As a technique for transforming Bacillus subtilis, any may be used as long as it is a technique now known in the art.

For example, the method proposed by Chang et al. can be used (Reference 24). This method can be divided into three steps as follows:

(1) Step of preparing cell wall-free Bacillus subtilis, i.e. a protoplast by treating Bacillus subtilis with lysozyme in an isotonic solution.

(2) Step of transformation of the protoplast with vector DNA in a polyethylene glycol solution.

(3) Step of reconstitution of the cell wall for the protoplast in a reproductive culture medium, and selecting the transformed Bacillus subtilis.

To obtain the hirudin using the transformed strain thus prepared, liquid cultivation of the strain by the usual method. For example, the transformed strain is inoculated into 400 ml of an LB culture medium (Reference Literature 25) in a 2-liter Erlenmeyer flask, and the cultivation is then carried out at 37ºC for about 20 hours with shaking, preferably until a maximum yield of hirudin has been secreted and produced.

The transformed strain of the present invention can be cultured in a liquid medium containing a digestible carbon source, nitrogen source and inorganic salt source. For example, the liquid culture medium that can be commonly used is an LB culture medium.

As the strain used in the present invention, any can be used as long as it is a Bacillus bacterium that can be transformed by the hirudin secretion plasmid of the present invention. Bacillus subtilis is the best because its data based on genetics, biochemistry, molecular biology and applied microbiology are numerous and the safety is high.

The production of hirudin from the culture medium can be achieved by reclaiming purification from the supernatant of the culture medium. The method of hirudin recovery found by the present inventors is as follows: The medium supernatant is adjusted to pH 3 with hydrochloric acid and the treatment is then carried out at 70°C for 15 minutes to form a protein precipitate. Subsequently, the latter is removed therefrom by centrifugation to obtain a supernatant fraction containing hirudin. In the supernatant fraction, hirudin is present in a high ratio of 20% with respect to the total protein, and it contains only a very small amount of impure proteins. Therefore, the hirudin secreted into the supernatant of the culture medium can be easily purified by cation exchange chromatography, Anion exchange chromatography and affinity chromatography.

The present inventors have found that hirudin can be secreted and enriched with an antithrombin activity corresponding to 10 mg/L x A660 by culturing Bacillus subtilis transformed with the hirudin secretion plasmid in a 2-fold concentrated LB culture medium. In this case, the absorbance (A660) of the culture medium indicating the growth stage of Bacillus subtilis is 10. The above-mentioned unit (mg/L x A660) shows a value obtained by dividing the amount (mg) of enriched hirudin in the medium supernatant (1 liter) by the absorbance (A660) indicating the growth stage of Bacillus subtilis in the culture medium.

It has been found that 35 mg of hirudin can be obtained from 1 liter of culture medium by adjusting the supernatant (1 liter) to pH 3 with hydrochloric acid, then treating at 70°C for 15 minutes to form a protein precipitate, removing the latter to obtain a supernatant fraction, and purifying the supernatant fraction by using anion exchange chromatography and affinity chromatography. The present invention has been achieved on the basis of this knowledge.

Furthermore, the hirudin secretion plasmid of the present invention has the DNA region in which the 5' terminus of the DNA fragment of hirudin has been directly ligated to a site immediately below the 3' terminus of the secretion signal region of the neutral protease gene, and it can be considered that in the bacteria or cytoplasmic membranes of the transformed strain obtained by carrying out the transformation with this plasmid, a precursor protein is synthesized in which an amino acid present at the N-terminus of hirudin is ligated to a site directly after an amino acid present at the C-terminus of the secretion signal of the neutral protease. It is known that the secretion signal is generally removed in the course of secretion, and also in the present invention, the secretion and production of such a natural type hirudin having the N-terminal amino acid sequence as in a secretion signal-free hirudin derived from Hirudo medicinalis can be expected. According to the report of Schein et al. (Reference 20) However, only by ligating the DNA fragment encoding a desired mature protein to a site immediately after the region encoding the promoter, ribosome binding region and secretion signal of the gene encoding a secretion protein, the mature protein from which the secretion signal has been removed cannot be efficiently secreted in a timely manner from the precursor protein, where the precursor protein is assumed to be synthesized in bacteria and in which the mature protein is fused to the C-terminus of the secretion signal. Also, in the case where Bacillus subtilis was transformed with the hirudin secretion plasmid of the present invention, it was undetermined whether or not the natural hirudin from which the secretion signal has been removed is secreted from the precursor protein in which an amino acid present at the N-terminus of hirudin is bound to a site immediately after an amino acid present at the C-terminus of the secretion signal of the neutral protease.

The present inventors have studied various ligation modes between the secretion signal and hirudin and as a result, they have found that only in the ligation mode of the present invention, the natural type hirudin is secreted into a culture medium by means of the transformed Bacillus subtilis as shown in the examples of the present case.

As shown in an embodiment of the present invention, hirudin having the same N-terminal amino acid sequence and the same amino acid composition as hirudin derived from Hirudo medicinalis can be secreted into the supernatant of a culture medium without any decomposition by using the promoter, ribosome binding region and secretion signal region of the neutral protease gene of Bacillus amyloliquefaciens to assemble a hirudin secretion plasmid, then introducing this plasmid into Bacillus subtilis to obtain a transformed strain and culturing the strain. That is, a hirudin preparation method has been established in which hirudin can be easily recovered and purified.

Now, the present invention will be described in detail with reference to the examples, but the scope of the present invention should not be limited thereto.

example 1 (Assembly of the heteroprotein expressing and secreting vector pNPA225)

A procedure shown in Figure 1 was carried out to assemble a heterologous protein expressing and secreting vector pNPA225 in which the DNA fragment encoding the heterologous protein was ligated to a site immediately after the region encoding the promoter, ribosome binding region and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens.

In Figure 1, plasmid pNPA84 is an amylase secretion plasmid having the DNA fragment (α-amylase) encoding the mature α-amylase protein at a site after the region encoding a promoter (Promoter), a ribosome binding region (SD), secretion signal (pre) and propeptide (Δpro) of the neutral protease gene. The mature α-amylase gene can be defined as the DNA fragment encoding a naturally occurring protein having α-amylase activity and starting with leucine. From the transformed strain MT-8400 (FERM BP-923) containing the plasmid pNPA84, the latter pNPA84 was prepared in accordance with the method of Tabak et al. (Reference 26). This pNPA84 DNA was then cleaved with restriction enzymes HpaII and BamHI (both of which were manufactured by Takara Shuzo Co., Ltd.) to obtain approximately 7.8 Kb of a DNA fragment (hereinafter referred to as "DNA fragment A"), and the latter was purified by electrophoresis using agarose gel. This DNA fragment contained the region encoding the promoter, the ribosome binding region and the C-terminus-free secretion signal. On the other hand, for the purpose of cleaving the neutral protease gene immediately after its secretion signal region with a restriction enzyme StuI, two kinds of oligonucleotides, i.e., 16mer and 18mer, were synthesized by an improved triester method (Reference Literature 27). Thereafter, 1 µg of each of the two kinds of synthesized oligonucleotides was phosphorylated using T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) and dATP (manufactured by Pharmacia Labs., Inc.) (Reference Literature 28). Next, these reaction products were mixed and then heated in hot water for 3 minutes, followed by slow cooling, whereby the two kinds of synthesized oligonucleotides were annealed. Thereafter, the DNA fragment (0.5 µg) and the annealed synthesized oligonucleotide (1 µg) were ligated to each other by using T4 ligase (manufactured by Takara Shuzo Co., Ltd.) to obtain a hybrid plasmid pNPA225.

Example 2 (Assembly of the hirudin secretion vector pNPH208)

A plasmid pNPH208 was assembled according to the procedure shown in Fig. 2.

First, for the purpose of assembling a DNA fragment (Fig. 3) containing the DNA region encoding an amino acid from the N-terminus to the 10th residue of hirudin, two kinds of oligonucleotides were synthesized. Next, 1 µg of each of the two kinds of oligonucleotides was phosphorylated and then annealed. This was ligated to pNPA225 DNA (0.5 µg) which had been digested with restriction enzymes StuI and BamHI by using T4 ligase (manufactured by Takara Shuzo Co., Ltd.) to obtain a hybrid plasmid pNPA225ΔH in which the N-terminal region (corresponding to the part from the N-terminus to the 10th amino acid residue of hirudin) of the DNA fragment encoding hirudin was ligated to a site immediately after the region encoding the secretion signal of the neutral protease gene.

A strain has already been deposited (FERM BP-1985) which was transformed with the plasmid p4014 assembled from the vector pBR322 and a synthesized DNA prepared by selecting a codon corresponding to the amino acid sequence of hirudin from codons commonly used in Bacillus subtilis, i.e., optimum codons, and then chemically synthesizing the codon. The DNA fragment (H) coding for hirudin was prepared from this transformed strain B9-1985 or BP-1985 in the following manner: First, p4014 was cleaved with restriction enzymes EcoRI and BamHI to prepare a DNA fragment containing hirudin. The DNA fragment thus prepared was placed between the cleavage sites of the plasmid pUC 13 with the restriction enzymes EcoRI and BamHI to assemble a hybrid plasmid p3009 (Fig. 4). This p3009 DNA (2 μg) was digested with the restriction enzymes AccIII and HindIII to prepare the DNA fragment (DNA fragment B) containing the DNA region encoding the amino acid sequence from the 11th amino acid to the C-terminus of hirudin, and the DNA fragment B was then purified by electrophoresis.

This DNA fragment B and pNPA225ΔH were digested with restriction enzymes AccIII and HindIII to produce approximately 6.0 Kb of a DNA fragment (DNA fragment C) (1 µg) and were ligated using T4 ligase to assemble a hirudin secretion vector pNPH208. This pNPH208 was a hybrid plasmid containing the DNA sequence in which the DNA fragment encoding hirudin had been ligated to a site immediately after the region encoding the promoter, ribosome binding region and secretion signal of the neutral protease gene of Bacillus amyloliquefaciens.

Example 3 (Secretion and production of hirudin by the hirudin secretion plasmid)

By using the plasmid pNPH208 assembled in Example 2, a Bacillus subtilis MT-430 strain (FERM BP-1079) was transformed in accordance with the method of Chang et al. (Reference Literature 24). The resulting transformed strain MT-208 (FERM BP-1983) was subjected to a shaking culture process at 37°C for 20 hours by using a 2-fold concentrated LB culture medium. The amount of secreted and accumulated hirudin was determined by measuring an antithrombin activity in the supernatant of the culture medium. This antithrombin activity was determined by measuring an inhibitory degree of the hydrolysis activity of a synthesized substrate HD-phenylanil-L-pipecolyl-L-arginyl-p-nitroanilide to thrombin (Reference Literature 30). That is, 50 μl of a thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) solution (20 IU/mL) was mixed with 50 μl of a buffer solution (50 mM Tris-HCl, pH=8.0) under neutral to acidic conditions, and the mixture was then incubated at room temperature for 2 minutes. Thereafter, 50 μl of the mixture was added to HD-phenylanil-L-pipecolyl-L-arginyl-p-nitroanilide (Daiichi Chemical Pharmaceutical Co., Ltd.) (final concentration of substrate = 0.25 mM). After the start of the reaction, the release of p-nitroanilide was measured at a wavelength of 405 nm, and the increase in absorbance per unit time was represented by a. Next, the buffer solution was replaced with a sample solution, and the same procedure was then carried out to measure the increase in absorbance at a wavelength of 405 nm. The measured value was represented by b. An antithrombin activity of the supernatant of the culture medium was determined by calculating (a - b)/a. One unit of antithrombin activity can be defined as the ability to neutralize one unit of 1N thrombin. The content of hirudin was determined by first separately measuring a thrombin specific activity and then by taking advantage of the fact that hirudin inhibits thrombin in a ratio of 1:1 (molar ratio).

After the cultivation was carried out at 37°C for 20 hours by using a 2-fold concentrated LB culture medium, an antithrombin activity of about 116 million units, that is, about 100 mg of enriched hirudin per liter of culture medium, was observed. In this case, the absorbance (A660) of the culture medium, which indicates the growth stage of Bacillus subtilis, was 10. Furthermore, the antithrombin activity of the secreted and enriched hirudin did not decrease, but it increased with time. When a precipitate obtained by adding trichloroacetic acid to the supernatant of the culture medium was analyzed by SDS-PAGE, was observed that a protein having the same size as a hirudin preparation obtained by purifying a hirudin sold by Sigma Chemicals Co. was accumulated in large amounts in the supernatant of the culture medium. In addition, according to the analytical results by a gel scanning densitometry, the amount of hirudin in the supernatant of an MT-208 strain culture medium was as much as 5%. When this supernatant was thermally treated at 70ºC for 15 minutes to form a proteinaceous precipitate and the latter was then removed therefrom by centrifugation, the antithrombin activity was still present in the supernatant fraction. Moreover, the ratio of existing hirudin protein to total protein was increased to as much as 20%, indicating that impure proteins were significantly reduced. Hirudin (Reference Literature 31) obtained by purifying this supernatant showed a band in SDS-PAGE, and it was seen that the molecular weight of this hirudin was the same as that of a hirudin preparation obtained by purifying a hirudin derived from Hirudo medicinalis commercially available from Sigma Chemicals Co. The N-terminal amino acid sequence of the purified hirudin was then measured, and as a result, it was determined that the sequence was Val-Val-Tyr-Thr-Asn.

The purified hirudin (10 mg) was hydrolyzed in 6N hydrochloric acid at 110°C for 24 hours and the hydrolyzed substance was then analyzed for its amino acid composition (Reference Literature 33). The results are shown in Table 1. It is clear that the analytical values in the table are closely consistent with the theoretical values of an amino acid composition calculated from the amino acid sequence of a hirudin derived from Hirudo medicinalis.

This fact indicates that hirudin produced by Bacillus subtilis infected with the hirudin secretion plasmid of the present invention has the same N-terminal amino acid sequence and the same amino acid composition as hirudin derived from Hirudo medicinalis.

As a result of the thrombin inhibition experiments, it became clear that this purified hirudin was able to react with thrombin stoichiometrically in a ratio of 1:1.14.

This fact also indicates that hirudin secreted and produced by Bacillus subtilis transformed with the hirudin secretion plasmid of the present invention has the same antithrombin activity as hirudin derived from Hirudo medicinalis. Table 1 Analytical results of amino acid composition Number of amino acids Theoretical value Found value

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Claims (8)

1. Hirudin secretion plasmid in which a DNA sequence has been ligated into a vector DNA, the DNA sequence having been prepared by ligating a DNA fragment encoding hirudin into a site immediately behind the region encoding the promoter, ribosome binding site and secretion signal of the neutral protease gene of a Bacillus bacterium. 2.Hirudin secretion plasmid according to claim 1, wherein the Bacillus bacterium is Bacillus amyloliquefaciens. 3.Hirudin secretion plasmid according to claim 1, wherein the DNA sequence is the following DNA sequence: 4.Hirudin secretion plasmid according to claim 1, wherein the vector DNA is a plasmid capable of being replicated in the genus Bacillus bacterium. 5.Hirudin secretion plasmid according to claim 1, wherein the plasmid that can be replicated by means of the Bacillus bacterium is pUB110. 6. A transformed strain which has been transformed with a hirudin secretion plasmid selected from the hirudin secretion plasmids described in claims 1 to 5. 7. Transformed strain according to claim 6, wherein a microorganism to be transformed is Bacillus subtilis. 8.A process for producing hirudin, the process comprising the steps of: culturing the strain described in claim 7 in a culture medium and then recovering hirudin from the supernatant of the culture medium.